CN105039362B - A kind of gene mutation improves antioxidative glucose oxidase and its method - Google Patents
A kind of gene mutation improves antioxidative glucose oxidase and its method Download PDFInfo
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Abstract
Antioxidative glucose oxidase is improved the invention proposes a kind of gene mutation and its method, the glucose oxidase are made of GOD-M524L-M528L mutated gene.Method are as follows: computer simulation analysis glucose oxidase sequence, design glucose oxidase encoding gene mutant are GOD-M524L-M528L；Full genome synthesizes the gene order, is expanded by Overlap extension PCR；It is cloned on pPIC9K carrier, electricity is transferred in Pichia pastoris GS115 bacterial strain after linearisation, it is screened again through culture expression, detection, shake flask fermentation collects fermented supernatant fluid, fermented supernatant fluid is dialysed and removes salt ion, pure enzyme sample is obtained through chromatographic purifying, is detected after oxidation processes 3.5h, the residual enzyme activity of control group GOD only has 20%, and the residual enzyme activity for being mutated glucose oxidase GOD-M524L-M528L then reaches 30%, improves its inoxidizability.
Description
Technical field
The present invention relates to molecular enzyme engineering technology, especially a kind of gene mutation improves antioxidative grape glycosyloxy
Change enzyme and its method.
Background technique
Glucose oxidase GOD is oxidoreducing enzyme (E1.1.3.4), and glucose is oxidized to grape by its energy specificity
Saccharic acid and hydrogen peroxide, according to the difference of reaction condition, reaction equation is as follows:
In the presence of no catalase, 1 mole of oxygen is consumed when every molar glucose oxidase.
C6H12O6+O2→C6H12O7+H2O2
In the presence of catalase, 0.5 mole of oxygen is consumed when every molar glucose oxidase.
C6H12O6+1/2O2→C6H12O7
In the case where having ethyl alcohol and catalase to exist simultaneously, hydrogen peroxide is used for the oxidation of ethyl alcohol simultaneously, at this point,
1 mole of oxygen is consumed when every molar glucose oxidase.
C6H12O6+C2H5OH+O2→C6H12O7+CH3CHO+H2O
Methionine (Met) is the one kind for constituting protein essential amino acid, must for protein methylation and a turn vulcanization
Need, methionine is easily oxidized, it vulnerable to hydrogen peroxide, hypochlorite, hydroxyl radical free radical oxidation and generate methionine
Sulfoxide, methionine sulfoxide, which is further oxided, can form methionine sulfone.Methionine sulfoxide can under given conditions may be used
To be reduced to methionine, and it is irreversible that methionine, which is oxidized to methionine sulfone in biology,.
H2O2It is considered as the competitive inhibitor of glucose oxidase GOD, the Michaelis constant and oxygen of Reverse transcriptase
It is about the same for the Michaelis constant of the inhibition of glucose oxidase GOD.Meanwhile reaction product H2O2To in glucose oxidase
The oxidation of Met and make its activity reduce or inactivation.Mutation glucose oxidase inoxidizability how is improved, makes it in grape glycosyloxy
It turns to and improves productivity in the production of gluconic acid, reduce production cost, there is great significance.
Summary of the invention
The purpose of the present invention is the method using gene mutation, rite-directed mutagenesis conventional glucose oxidizing ferment improves mutation Portugal
Grape carbohydrate oxidase inoxidizability makes it improve productivity in the production that glucose is oxidized to gluconic acid, reduces production cost.
The invention is realized in this way being designed by computer-assisted analysis, rite-directed mutagenesis glucose oxidase (GOD) is special
Methionine is replaced with leucine, and it is expressed in Pichia pastoris by anchor point methionine, filters out raising mutation Portugal
Grape carbohydrate oxidase inoxidizability makes it improve productivity in the production that glucose is oxidized to gluconic acid, reduces production cost.
The described gene mutation improves antioxidative glucose oxidase by GOD-M524L-M528L mutated gene structure
At, i.e., by its gene order 524 and 528 MET amino acid mutations be LEU.
1. computer simulation analysis glucose oxidase sequence and molecular docking
By analyzing (such as Fig. 1) to glucose oxidase sequence, the flavin adenine dinucleotide (FAD) of GOD of the present invention
The corresponding amino acid residue in combined area (flavin adenine dinucleotide, FAD) (underscore mark) be 16-54,
239-306,529-553,560-583, FAD combination expansion area (box mark) corresponding amino acid residue be Y68, R230,
The corresponding amino acid residue of C521, the FAD area of coverage (wave mark) is 76-96, and flavine attachment band (double underline mark) is right
The amino acid residue answered is 97-114.This experiment all avoids these binding sites when designing Amino Acid-Induced Site-Directed Mutation below as far as possible.
On the basis of to glucose oxidase sequential analysis of protein, three of design glucose oxidase encoding gene are prominent
Variant is GOD-8, GOD-M523L-M524L (see sequence table NO2) and GOD-M524L-M528L (see sequence table NO3), passes through meter
Three mutant are carried out molecular docking with substrate glucose respectively by calculation machine three mutant structures of homologous modeling assistant analysis
(based on CDOCKER docking analysis software), to study active influence after GOD mutation.
2. glucose oxidase and the building for being mutated body expression vector
Pass through full genome synthesis mutant GOD-8 gene order；And GOD-M523L- is expanded by Overlap extension PCR
M524L, GOD-M524L-M528L mutant gene sequence, specific as follows:
GOD-M523L-M524L by two-wheeled PCR amplification, mutational site design primer be GOD-M1-F, GOD-M1-R,
GOD-F-EcoR I and GOD-R-Not I.
GOD-M524L-M528L by two-wheeled PCR amplification, mutational site design primer be GOD-M2-F, GOD-M1-R,
GOD-F-EcoR I and GOD-R-Not I.
3. expression, screening and purifying in Pichia pastoris
After synthesis mutant gene, by the way that on molecular cloning to pPIC9K carrier, recombinant expression carrier is linearized with Sal I
Electricity is transferred in Pichia pastoris GS115 bacterial strain afterwards, and 28 DEG C are cultivated 3~4 days, and the recon that will be grown on MD plate is transferred simultaneously
Onto MD and MM plate, 28 DEG C of incubator culture 48h.By developing solution (by 10mL containing 1% agarose deionized water heating dissolves,
It is to be cooled to 60 DEG C hereinafter, immediately be added 2mL18%D- glucose, 200 μ L1% be dissolved in methanol dianisidine solution and
400 μ L 90U/mL horseradish peroxidases mix, and mixture is developing solution) it is poured on master plate to be checked, it is stored at room temperature
It develops the color, after the 30min that develops the color, expression of the GOD in Pichiapastoris expression strain is judged according to colour developing circle size.It will screen
To the higher bacterial strain of expression activity, shaking flask is expressed in BMGY and BMMY culture medium, after shaking flask 120h, collects fermented supernatant fluid.It will
Fermented supernatant fluid dialysis removal salt ion in 0.05mM Tris-HCL (PH8.0).It is obtained through Cellulose DE column chromatography
To pure enzyme sample.
The BMGY culture medium are as follows: 1% yeast extract, 2% peptone, 1.5% agar, 121 DEG C sterilize 20 minutes
Afterwards, addition 1.34%YNB, 4 × 10-5% biotin, 100mM pH6.0 phosphate buffer, 1% glycerol
The BMMY culture medium are as follows: 1% yeast extract, 2% peptone, 1.5% agar, 121 DEG C sterilize 20 minutes
Afterwards, addition 1.34%YNB, 4 × 10-5% biotin, 100mM pH6.0 phosphate buffer, 0.5% methanol
4. the measurement of enzymatic activity and protein content
GOD survey enzyme activity principle: under the action of glucose oxidase, glucose and oxygen reaction, generate gluconic acid and
Hydrogen peroxide, hydrogen peroxide and colourless reduced form dianisidine generate water and red under the action of peroxidase
Oxidized o-Dianisidine.
The definition of GOD enzyme-activity unit: at 37 DEG C, under conditions of pH6.0, D- is oxidized to from β-D-Glucose of 1 μm of ol within 1 minute
Gluconic acid and H2O2Required enzyme amount is a glucose oxidase enzyme activity unit, is indicated with U/g (U/ml).
The drafting of standard curve:
Glucose oxidase standard items are selected, various concentration of glucose oxidizing ferment standard solution are prepared.Use phosphate-buffered
Liquid is diluted to 0.8U/mL, 1.2U/mL, 1.6U/mL, 2.0U/mL, 2.4U/mL, 2.8U/mL.It takes in 10ml test tube, is added
2.5ml dianisidine, 0.3ml glucose solution, 0.1ml Catalase solution anabolic reaction system, reaction system is 37
After DEG C heat preservation 5min, it is separately added into various concentration of glucose oxidizing ferment standard solution 0.1mL, after reacting 3min, 2M H is added2SO4
(concentrated sulfuric acid: water v/v=1:8) 2mL, oscillation shake up to terminate reaction, measure light absorption value using spectrophotometer 540nm wavelength.
It is X-axis by Y-axis, absorbance OD value of glucose oxidase concentration, draws standard curve.
Determination step: appropriate diluted enzyme solution (extension rate is determined according to specific concentration) is drawn, starts to operate on.Benefit
The concentration of glucose oxidase in test specimen is calculated with glucose oxidase standard curve.
The calculating of sample enzyme activity:
Y=[K (AE-AB)+C0] × D
Activity of glucose oxidase in Y-sample dilution, U/mL；AE-enzyme reaction solution absorbance；
AB-enzyme blank sample absorbance；K-standard curve slope；
CO-standard curve intercept；The total extension rate of D-sample.
The present invention surveys protein concentration using Bradford method.The drafting of standard curve: standard protein liquid is diluted to 10 μ
The protein concentration gradient solution of g/ml, 20 μ g/ml, 40 μ g/ml, 60 μ g/ml, 80 μ g/ml and 100 μ g/ml, take 1ml to put respectively
Enter in 10ml test tube, 5ml Coomassie brilliant blue reagent is added in every pipe, mixes, and static 5min measures its absorbance in 595nm, with egg
White concentration is abscissa, using absorbance as ordinate, draws standard curve.
The measurement of soluble protein: taking 1ml sample to be tested to be put into 10ml test tube, and 5ml Coomassie brilliant blue reagent is added in every pipe,
It mixes, static 5min measures its absorbance in 595nm, the concentration of its soluble protein is calculated by standard curve.
5. glucose oxidase inoxidizability measures
GOD sample 4ml to be measured is taken, 1ml hydrogen peroxide is added, 37 DEG C of heat preservation 3.5h take a sample every 0.5h, at once
Excess hydrogen peroxide enzyme (400u/ μ L) is added and removes extra hydrogen peroxide, measurement residual enzyme activity.
The present invention devises three kinds of different mutants of glucose oxidase by computer-assisted analysis, and red by finishing
Yeast expression obtains corresponding pure enzyme, and property analysis shows that one of mutant inoxidizability significantly improves.The method avoids
The experiment blindness of random mutation can effectively improve the efficiency of enzyme molecule transformation.The method is also suitable for other enzyme molecules
The transformation of matter.
Detailed description of the invention
The supposition of Fig. 1, GOD amino acid sequence and binding site
Fig. 2, GOD and D-Glucose interaction 2 d plane picture
Fig. 3, GOD-8 and D-Glucose interaction 2 d plane picture
The detection of Fig. 4, GS115-pPic9K-GOD-8 plate
Fig. 5, GOD-M523L-M524L and substrate interaction 2 d plane picture
Fig. 6, GS115-pPic9K-GOD plate screening
Fig. 7, GS115-pPic9K-GOD-M523L-M524L plate screening
Fig. 8, GOD-M524L-M528L and substrate interaction 2 d plane picture
Fig. 9, GS115-pPic9K-GOD-M524L-M528L plate screening
Specific embodiment:
Embodiment 1:GOD and the analysis of mutant GOD-8 data
10 pose are shared after GOD and substrate D-Glucose CDOCKER docking, wherein-the CDOCKER of optimal pose
Energy is that 5.07234, CDOCKER interaction energy is 26.6475 (such as Fig. 2).By invention specific implementation step
Pichia anomala expression is carried out, the GOD enzyme activity measured after purification is 442.755U/ml, and soluble protein is 136.476 μ g/ml, than work
Power is 3.24U/ μ g.
10 pose are shared after GOD-8 and substrate D-Glucose CDOCKER docking, wherein-the CDOCKER of optimal pose
Energy is that 6.77139, CDOCKER interaction energy is 30.6795 (such as Fig. 3).By invention specific implementation step
Pichia anomala expression is carried out, is developed the color and is screened by plate, discovery mutant GOD-8 is without active (such as Fig. 4).
Embodiment 2: mutant GOD-M523L-M524L data analysis
10 pose are shared after GOD-M523L-M524L and substrate D-Glucose CDOCKER docking, wherein optimal pose
- CDOCKER energy be 0.242621 ,-CDOCKER interaction energy be 25.579 (such as Fig. 5).By invention
Specific implementation step carries out Pichia anomala expression, is developed the color and is screened by plate, and mutant GOD-M523L-M524L activity is (as schemed
7) lower than wild type GOD (such as Fig. 6), the enzyme activity of mutant GOD-M523L-M524L purifying is 1.210U/ml, and soluble protein is
26.476 μ g/ml, Rate activity are 0.0457U/ μ g, because mutant enzyme work is very low thus, are surveyed not after by hydrogen peroxide treatment
Enzyme activity out, so not comparing the inoxidizability of itself and GOD.
Embodiment 3: mutant GOD-M524L-M528L data analysis
10 pose are shared after GOD-M524L-M528L and substrate D-Glucose CDOCKER docking, wherein optimal pose
CDOCKER energy is that 0.877411, CDOCKER interaction energy is 23.5475 (such as Fig. 8).Have by invention
Body implementation steps carry out Pichia anomala expression, are developed the color and are screened by plate, mutant GOD-M524L-M528L (such as Fig. 9) activity
It is higher than wild type GOD.The enzyme activity of mutant GOD-M524L-M528L purifying is 245.843/ml, and soluble protein is 48.476 μ g/
Ml, Rate activity are 3.59U/ μ g.By GOD and GOD-M524L-M528L by after hydrogen peroxide treatment 3.5h, as grape is glycoxidative
The increase for the time that enzyme is incubated in hydrogen peroxide, the inoxidizability of GOD-M524L-M528L is always to be higher than GOD, by mistake
After hydrogen oxide handles 3.5h, the residual enzyme activity of GOD only has 20%, and the residual enzyme activity of GOD-M524L-M528L then reaches 30%,
Illustrate that by 524 and 528 MET amino acid mutations be LEU, its inoxidizability can be improved.
Claims (1)
1. a kind of gene mutation improves antioxidative glucose oxidase, it is characterised in that the gene mutation improves inoxidizability
Glucose oxidase be made of GOD-M524L-M528L mutated gene, i.e., by its gene order 524 and 528 MET ammonia
Base acid mutation is LEU, and particular sequence is sequence table NO3: glucose oxidase mutant GOD-M524L-M528L gene sequence
Column.
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| CN105463062B (en) * | 2016-01-21 | 2018-06-29 | 山东大学 | A kind of tablet coloration method of quick detection filamentous fungi synthesis of oligonucleotides fructose ability |
| CN106636021A (en) * | 2016-12-14 | 2017-05-10 | 曹书华 | Method for improving oxidation resistance of glucose oxidase |
| CN111349622B (en) * | 2018-12-20 | 2022-04-08 | 南京百斯杰生物工程有限公司 | Glucose oxidase mutant and application thereof in industrial production |
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